Matrix Metalloproteinases (MMPs) are a family of over 20 types of enzymes, both secreted and membrane-bound zinc endopeptidases, which collectively are capable of degrading all the components of the extracellular matrix. Studies suggest that MMPs are utilized in cancer, and facilitate both local tumor invasion and metastasis. MMP-2 and MMP-9 (also known as gelatinases), in particular, are thought to play critical roles in tumor cell invasion and are frequently coexpressed in human cancers. The goal of this study is to test a hypothesis regarding the ability to image by positron emission tomography (PET) the expression of MMP-2 and MMP-9 in a metastatic breast cancer tumor model using radiolabeled MMP-2 and MMP-9 peptide inhibitors. The hypothesis to be tested is that the accumulation of a radiolabeled gelatinase inhibitor will correlate with gelatinase enzyme activity determined in the tumor in an ex vivo assay. It has been demonstrated by Koivunen et al. that the synthetic cyclic peptide, CTTHWGFTLC (CTT), suppressed migration of breast cancer tumor cells in vitro and prevented the growth and invasion of MDA-MB-435 human breast cancer tumors in mice, suggesting that this peptide has potential as an anticancer agent. In preliminary experiments relating to this proposal, we conjugated CTT with the chelator DOTA (1,4,7, 10-tetraazacyclotetradecane-N,N',N",N'"- tetraacetic acid) and initial in vitro assays demonstrated that DOTA-CTT inhibits MMP-2 activity comparably to the broad-range MMP inhibitor, Ilomastat. Preliminary microPET imaging and biodistribution studies in tumor-bearing mice show 64Cu-DOTA-CTT is taken up in three histologically different tumor types. Our preliminary data show that a 64Cu-labeled negative control peptide, DOTA-D-Trp-CTT does not show significant tumor uptake. Our specific aims are as follows: 1) to optimize fluorogenic assays for determining inhibitory activity of MMP-2 and MMP-9 inhibitors and for determining MMP-2 and MMP-9 concentrations in tumors grown in vivo; 2) To confirm specific tumor uptake of 64Cu-DOTA-CTT vs a 64Cu-DOTA-conjngated control peptide in gelatinase-expressing tumor-bearing mouse models from biodistribution studies and/or microPET imaging data and to correlate uptake of 64Cu-DOTA-CTT with gelatinase expression in the tumor using an ex vivo assay; and 3) to synthesis a series of gelatinase inhibitors based upon the lead structure CTT that are conjugated to either DOTA or a cross-bridged macrocyclic chelator, as well as synthesize negative control peptides. If successful, the outcome of this study may be a PET tracer for determining the metastatic potential of various types of cancer.